This invention is in the field of solid-phase organic synthesis, and specifically relates to a method of linking complex and sterically demanding hydroxylic compounds to a solid support for subsequent synthetic transformation and polymer-linked compositions obtained thereby. The method is especially suited to the preparation of complex carbohydrates and glycoconjugates such as glycopeptides.
New methods of preparing complex glycoconjugates are needed in view of the growing recognition of the role played by complex carbohydrates as signaling molecules in the context of biological processes has recently gained prominence. M. L. Phillips, et al., Science, 1990, 250, 1130; M. J. Polley, et al., Proc. Natl. Acad. Sci. USA, 1991 88, 6224: T. Taki, et al., J. Biol. Chem., 1996, 261, 3075). For example, the carbohydrate domains of the blood group substances contained in both glycoproteins and glycolipids are distributed in erythrocytes, epithelial cells and various secretions. Such determinants are encountered as markers for the onset of various tumors. K. O. Lloyd, Am. J. Clinical Path., 1987, 87, 129; K. O. Lloyd, Cancer Biol., 1991, 2, 421. Carbohydrate-based tumor antigenic factors have applications at the diagnostic level, as resources in drug delivery or ideally in immunotherapy. Toyokuni, T., et al., J. Am. Chem Soc. 1994, 116, 395; Dranoff, G., et al., Proc. Natl. Acad. Sci. USA 1993, 90, 3539; Tao, M-H.; Levy, R., Nature 1993, 362, 755; Boon, T., Int. J. Cancer 1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol. 1992, 4, 624; Hakomori, S., Annu. Rev. Immunol. 1984, 2, 103; K. Shigeta, et al., J. Biol. Chem. 1987, 262, 1358.
Antigens which are selective (or ideally specific) for cancer cells could prove useful in fostering active immunity. Hakomori, S., Cancer Res., 1985, 45, 2405-2414; Feizi, T., Cancer Surveys 1985, 4, 245-269. Novel carbohydrate patterns are often presented by transformed cells as either cell surface glycoproteins or as membrane-anchored glycolipids. In principle, well chosen synthetic glycoconjugates which stimulate antibody production could confer active immunity against cancers which present equivalent structure types on their cell surfaces. Dennis, J., Oxford Clycostems Glyconews, Second Ed., 1992; Lloyd, K. O., in Specific Immunotherapy of Cancer with Vaccines, 1993, New York Academy of Sciences, pp.50-58. Chances for successful therapy improve with increasing restriction of the antigen to the target cell. For example, one such specific antigen is the glycosphingolipid isolated by Hakomori and collaborators from the breast cancer cell line MCF-7 and immunocharacterized by monoclonal antibody MBrl. Bremer, E. G., et al., J. Biol. Chem. 1984, 259, 14773-14777; Menard, S., et al., Cancer Res. 1983, 43, 1295-1300.
An improved strategy for linking carbohydrates to solid supports would facilitate glycopeptide synthesis. Such a strategy would aid in the assembly of complex glycoconjugates, which require a high degree of convergence and relief from the burdens associated with the manipulation of blocking groups. An improved linking method would also allow greater flexibility in the conjugation of the carbohydrate determinant with carrier proteins or lipids. Bernstein, M. A.; Hall, L. D., Carbohydr. Res. 1980, 78, Cl; Lemieux, R. U., Chem. Soc. Rev. 1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc. 1975, 97, 4076. This is a critical condition if the synthetically derived carbohydrates are to be incorporated into carriers suitable for biological application.
Accordingly, the subject invention provides a novel approach to the synthesis of complex carbohydrates on polymer supports. In particular, the invention provides a new and generally applicable substrate-linking procedure for use in a wide variety of systems, including not only glycoconjugates but other complex organic systems containing at least one hydroxyl group. Roberge, J. Y. et al., Science, 1995, 269, 202; Randolph, J. T. et al., J. Am. Chem. Soc. 1995, 117, 5712; Danishefsky, S. J. et al., Science, 1993, 260, 1307. Prior solid state methods of glycopeptide synthesis suffer from the limitation that the initial silylation event which serves to xe2x80x9cloadxe2x80x9d the polymer provides poor coupling efficiencies when the loading is conducted at positions which bear relatively hindered glycoxyl centers.
The present methods have generated glycoconjugates not available by prior art methods. Huang, W-Q. J. Chem. Soc., Chem. Commun. 1985, 909. In the present method, even sterically encumbered alcohols can be attached to a solid support with little or no prior manipulation of a commercially available polymer, e.g., hydroxymethyl-polystyrene. The process requires only inexpensive materials and the linker is compatible with a variety of reaction conditions. Furthermore, the present method is applicable in an automatic glycal-based carbohydrate synthesizer, as the linker can be cleaved quickly without damaging the original polymer support (FIG. 1(b)). As a result, the solid support is then immediately ready for reloading, thereby providing an important economic advantage over prior methods.
One object of the present invention is to provide a method of linking complex hydroxylic compounds to a solid polymeric support. Specifically, the method disclosed herein uses a silylene linker to attach a hydroxylic compound to the support.
A particular object of the invention is to provide a polymer-linked composition having the structure: 
wherein RA and RB are each independently a C1-6 linear or C3-10 branched chain alkyl or an aryl group; wherein  is a polymeric support; wherein L is a linker selected from the group consisting of a single bond; a saturated or unsaturated oligomethylene chain, said oligomethylene chain being optionally interrupted by oxygen or sulfur atoms, an alkyl- or arylamino moiety, NHxe2x80x94CO or COxe2x80x94NH; a 1,4-phenylene; or a 1,4-phenylenemethylene moiety, said moiety being optionally substituted by at least one linear or branched alkyl or alkoxy group and/or halogen atom; and wherein RC is a linear or branched acyclic, cyclic or multicyclic moiety, said moiety being optionally unsaturated and/or substituted by at least one hydrogen, ORi, amino, alkyl- or dialkylamino, NHCORi, halogen, CH2OH, CH2ORi, a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein Ri is hydrogen, CHO, COORii, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group aryl, alkylaryl or arylalkyl; wherein if RC is cyclic, said moiety is optionally aromatic and/or heterocyclic; or if multicyclic, said moiety is optionally a fused multicyclic, fully or partially aromatic and/or heterocyclic.
A further object of the invention is to provide a method for preparing a polymer-linked composition having the structure: 
wherein RA and RB are each independently a C1-6 linear or C3-10 branched chain alkyl or an aryl group; wherein  is a polymeric support; wherein L is a linker selected from the group consisting of a single bond; a saturated or unsaturated oligomethylene chain, said oligomethylene chain being optionally interrupted by oxygen or sulfur atoms, an alkyl- or arylamino moiety, NHxe2x80x94CO or COxe2x80x94NH; a 1,4-phenylene; or a 1,4-phenylenemethylene moiety, said moiety being optionally substituted by at least one linear or branched alkyl or alkoxy group and/or halogen atom; and wherein RC is a linear or branched acyclic, cyclic or multicyclic moiety, said moiety being optionally unsaturated and/or substituted by at least one hydrogen, ORi, amino, alkyl- or dialkylamino, NHCORi, halogen, CH2OH, CH2ORi, a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein Ri is hydrogen, CHO, COORii, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group aryl, alkylaryl or arylalkyl; wherein if RC is cyclic, said moiety is optionally aromatic and/or heterocyclic; or if multicyclic, said moiety is optionally a fused multicyclic, fully or partially aromatic and/or heterocyclic; which comprises: (a) O-silylating HOxe2x80x94RC with X2SiRARB, wherein X is a halogen, in the presence of a base under conditions to form a halosilyl compound having the structure: XSiRARBxe2x80x94ORC; and (b) coupling the halosilyl compound formed in step (a) with a polymeric alcohol support in the presence of a base under conditions to form the polymer-linked composition.
Another object of the present invention is to provide a method of preparing a compound having the structure RCxe2x80x94OH wherein RC is a linear or branched acyclic, cyclic or multicyclic moiety, said moiety being optionally unsaturated and/or substituted by at least one hydrogen, ORi, amino, alkyl- or dialkylamino, NHCORi, halogen, CH2OH, CH2ORi, a substituted or unsubstituted linear or branched chain alkyl, (mono-, di- or tri)hydroxyalkyl, (mono-, di- or tri)acyloxyalkyl, arylalkyl or aryl group; wherein Ri is hydrogen, CHO, COORii, or a substituted or unsubstituted linear or branched chain alkyl, arylalkyl or aryl group aryl, alkylaryl or arylalkyl; wherein if RC is cyclic, said moiety is optionally aromatic and/or heterocyclic; or if multicyclic, said moiety is optionally a fused multicyclic, fully or partially aromatic and/or heterocyclic; which method comprises:
(a) preparing a polymer-linked composition having the structure: 
wherein RA and RB are each independently a C1-6 linear or C3-10 branched chain alkyl or an aryl group; wherein  is a polymeric support; wherein L is a linker selected from the group consisting of a single bond; a saturated or unsaturated oligomethylene chain, said oligomethylene chain being optionally interrupted by oxygen or sulfur atoms, an alkyl- or arylamino moiety, NHxe2x80x94CO or COxe2x80x94NH; a 1,4-phenylene; or a 1,4-phenylenemethylene moiety, said moiety being optionally substituted by at least one linear or branched alkyl or alkoxy group and/or halogen atom; and (b) treating the polymer-linked composition with a cleaving reagent under suitable conditions to form RCxe2x80x94OH.